Serum human chorionic gonadotropin ratios for the detection of etoposide, methotrexate, dactinomycin, cyclophosphamide, and vincristine resistance in high‐risk gestational trophoblastic neoplasia

Abstract Aims This study aimed to identify the optimal human chorionic gonadotropin (hCG) ratio in predicting etoposide, methotrexate, dactinomycin, cyclophosphamide, and vincristine resistance in women diagnosed with high‐risk gestational trophoblastic neoplasia (GTN) and to compare the chemoresistant disease detection rate by using the optimal hCG ratio and traditional criteria. Methods Seventy‐six women with primary high‐risk GTN treated with etoposide, methotrexate, dactinomycin, cyclophosphamide, and vincristine in a tertiary‐care center were included. The hCG ratio was determined by its serum pretreatment level divided by that before each cycle of chemotherapy. The traditional criteria for chemoresistance included plateau or rising of hCG or presence of new metastasis. The optimal hCG ratio was determined using receiver operating characteristics (ROC) curve analysis. Results Among the specificities of 90%, 92.5%, and 95%, the 90% specificity yielded the best ROC curve. At 90% specificity, the best area under curve value was at the fourth cycle with 75% sensitivity. The hCG ratio at the fourth cycle was 31.92. Using the ratio at the fourth cycle, chemoresistant disease was detected in six out of eight patients, compared to one in the traditional criteria. When combining the two diagnostic tools, the cumulative detection rate in the fourth cycle was 10/12 (83.3%) of total drug resistance. Among patients who developed drug resistance at the fourth cycle or thereafter, the use of the ratio at the fourth cycle could diagnose chemoresistance approximately two cycles earlier than that with the traditional criteria. Conclusions A hCG ratio of <31.9 at the fourth cycle should be considered a high‐risk for etoposide, methotrexate, dactinomycin, cyclophosphamide, and vincristine resistance and may need second‐line chemotherapy. The ratio increases the detection rate of resistance to these drugs more than the traditional criteria.


| INTRODUCTION
Gestational trophoblastic neoplasia (GTN) is a group of malignant neoplasms, including invasive mole, choriocarcinoma, placental-site trophoblastic tumor (PSTT), and epithelioid trophoblastic tumor (ETT), that arises from abnormal proliferation of placental trophoblast cells during pregnancy. GTN is highly sensitive to chemotherapy with a cure rate of >90%. 1,2 The serum human chorionic gonadotropin (hCG) level is a reliable tumor marker for diagnosis and monitoring response to treatment.
Patients with GTN are classified into low-risk (LRGTN) or high-risk (HRGTN) using the modified World Health Organization (WHO) prognostic scoring system as adapted by the International Federation of Gynecology and Obstetrics (FIGO). 3 HRGTN is defined by FIGO stage IV or any FIGO stage with a score of ≥7 which is unlikely to respond with single-agent chemotherapy treatment. Therefore, the standard treatment of HRGTN is combined chemotherapy. Etoposide, methotrexate, and dactinomycin alternated weekly with cyclophosphamide and vincristine (EMA/CO) regimen is effective, less toxic, and widely used as the primary treatment in HRGTN patients. 4,5 The remission rate of EMA/CO therapy ranges from 71-86%. [6][7][8][9][10] The patients who were refractory to EMA/CO had worse outcomes with only 43% 5-year survival (95% confidence interval: 12-73%). 11 The early diagnosis of chemoresistance can reduce unnecessary chemotherapy and chemotoxicity and may potentially improve the treatment outcomes. The diagnosis of EMA/CO resistance depends on a declining level of hCG. Currently, there is no consensus on the guidelines to define the criteria to determine drug resistance. A variety of tools derived from declining hCG levels were proposed to predict clinical course and response to chemotherapy such as hCG regression curve with or without a cut-off point, hCG ratio, and hCG reduction rate. [12][13][14][15][16] The utility hCG ratio in GTN was first introduced by Van Trommel et al. 17 in 2008 to identify persistent trophoblastic disease. 18 However, no study reports the use of hCG ratio to assess chemotherapy response in GTN. The current study aimed to evaluate the optimal serum hCG concentration ratios in primary HRGTN for predicting EMA/CO resistance. The secondary objective was to compare the detection rate of chemoresistant disease between optimal hCG ratio and traditional criteria.

| METHODS
Following approval by the research ethics committee, the medical records of patients with primary HRGTN who received the EMA/CO chemotherapy regimen between January 1, 2002, and December 31, 2013, were retrospectively reviewed. The requirement for the acquisition of informed consent from patients was waived owing to the retrospective nature of this study. Primary HRGTN was defined as patients who had initial FIGO stage IV or any stage with WHO scoring ≥7. Patients with recurrent disease, PSTT, ETT, discontinued treatment due to drug toxicity, or lost to follow-up and response could not be determined were excluded. Patient characteristics were reviewed for age, antecedent pregnancy, interval months from index pregnancy, size, and site of metastasis, pretreatment serum hCG level, and level before each cycle of chemotherapy, FIGO-2000 stage, and modified WHO risk-factor scoring system for GTN.
All primary HRGTN cases were evaluated for hCG ratios. The hCG ratio was defined as pretreatment serum hCG level divided by hCG level before each cycle of chemotherapy. The hCG ratios of each cycle were investigated by the area under the curve (AUC) from the receiver operating characteristics method (ROC) at 90%, 92.5%, and 95% specificities in predicting chemoresistance classified by the traditional criteria. The best cut-off hCG ratio was identified.
The detection of chemoresistance by using the optimal hCG ratio and the traditional criteria was compared. Cox regression analysis was performed for the univariate and multivariate analyses to identify independent risk factors associated with chemoresistance.
Complete response was defined when hCG regressed to a normal level for 3 consecutive weeks. Patients who had not achieved a complete response following the traditional criteria were considered chemoresistant: (i) plateau of hCG throughout three cycles of chemotherapy, (ii) rise in hCG of ≥10% over two cycles of chemotherapy, or (iii) presence of new metastasis.

| Immunoassays
The serum hCG levels were determined using MODULAR ANALY-

| Statistical analyses
From the sample size, calculations for diagnostic tests were based on 80% sensitivity, 95% specificity, 28% prevalence of EMA/CO resistance, 10 and 0.05 alpha level. At least 101 patients were required.
Statistical calculations were performed using R software version 2.14 (The R Foundation for Statistical Computing). Differences in the numerical data between the two groups were tested nonparametrically (Mann-Whitney U test) and parametrically (the Student t test). All tests were considered statistically significant at p < 0.05.

| Ethical approval
This retrospective cohort study was conducted in accordance with the principles embodied in the 1975 Helsinki Declaration, as revised in 2000, and the study protocol was approved by the Institutional  F I G U R E 1 Receiver operating characteristic curve of specificity of 90%, 92.5%, and 95% at the fourth cycle of therapy (optimal cut-point data)

| DISCUSSION
Early detection of drug resistance is beneficial in decreasing the number of cycles and toxicity from chemotherapy. The present study evaluated the optimal serum hCG concentration ratios in HRGTN cases receiving EMA/CO to predict chemotherapy resistance and found that the best cut-off point of hCG level for the diagnosis of chemoresistance is in the fourth cycle with a sensitivity and specificity of 70% and 90%, respectively. The AUC for hCG ratio to predict EMA/CO resistance demonstrates moderate discriminatory power (0.83); therefore, it has the potential utility as a diagnostic test. The hCG cut-off ratio in the fourth cycle is an independent risk factor for EMA/CO resistance and is considered a new tool to diagnose chemoresistance with a higher detection rate than the traditional criteria.
The clinical utility of declining hCG levels that is proposed to predict chemotherapy response in GTN is shown in  19 suggested that the best cut-off value of hCG was after third cycle, which could predict methotrexate resistance in LRGTN.
To the best of our knowledge, this is the first study to analyse the hCG ratios in HRGTN for EMA/CO chemoresistance. Lybol et al. 16 and Rattanaburi et al. 15 introduced the hCG regression nomogram in HRGTN to predict EMA/CO resistance. 15,16 Rattanaburi et al. 15 have discussed hCG regression curve for nomogram and reported that 90th percentile of the hCG level turned to normal before the eighth cycle of chemotherapy in primary HRGTN and found that a serum hCG level of >118.6 at the fifth cycle of chemotherapy predicted EMA/CO resistance with a sensitivity 85.7% and specificity of 100%. 15 Comparing the detection rate of the hCG ratio in our study to the hCG regression curve with cut-off value, it was found that the detection rate of hCG ratio had a lower sensitivity and specificity; T A B L E 3 Univariate and multivariate cox regression analyses for EMA/CO resistance in high-risk gestational trophoblastic neoplasia however the use of hCG ratio had the advantage of being able to detect chemoresistance at one cycle earlier.
Regarding clinical applications, the detection rate and timing for the diagnosis of chemoresistance were compared to those of the traditional criteria. Using the hCG ratio at the fourth cycle of chemotherapy could detect more chemoresistant diseases than that with the traditional criteria. On combining the two diagnostic tools, the cumulative detection rate at fourth cycle increased to 83% of total drug resistance.
Approximately half of the chemoresistant patients can be determined by the hCG ratio earlier than that with the traditional criteria. However, using the hCG ratio alone could not detect early chemoresistance that develops before the fourth cycle of chemotherapy which was found in approximately one-third of total chemoresistant cases in our series.
In agreement with the previous studies, we analysed hCG ratios at high specificity levels. The previous nomograms for the prediction of resistance to methotrexate chemotherapy used upper percentiles of p95, p97.5, and p99. 18,20 The study of using population kinetic modeling of hCG measurement for detection of methotrexate resistance accepted the specificity at 83% as the best cut-off point. 19 Our study used the specificities of only >90% to avoid false positive rates which cause unnecessary change to more intensive chemotherapeutic regimens. We did not include GTN patients with single chemotherapy resistance due to having a difference in hCG regression curve and different prognosis. 15 Our study has some limitations. Due to rarity of disease, the small Boonyapipat had full access to all of the data in this study and takes complete responsibility for the integrity of the data and the accuracy of the data analysis.